Oligonucleotides or polynucleotides immobilized on planar substrates are increasingly useful as diagnostic or screening tools. Polynucleotide arrays include regions of usually different sequence oligonucleotides or polynucleotides arranged in a predetermined configuration on the substrate. These regions (sometimes referenced as “features”) are positioned at respective locations (“addresses”) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example, all polynucleotide targets (e.g., DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array can be accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Polynucleotide arrays can be fabricated by depositing previously obtained polynucleotides onto a substrate, or by in situ synthesis methods. Various chemical schemes have been described for the synthesis of polynucleotides. Typically these methods use a nucleoside reagent of the formula:
in which:
A represents H or an optionally protected hydroxyl group;
B is a purine or pyrimidine base whose exocyclic amine functional group is optionally protected;
one of M or Q is a conventional protecting group for the 3′ or 5′-OH functional group (or, optionally, a conventional 3′ or 5′-OH protecting group at the end of an intervening (and optionally protected) polynucleotide sequence, e.g., such that formula (I) can represent a modified polynucleotide) while the other is:

where x can be 0 or 1, provided that:
a) when x=1:    R′ represents H and R″ represents a negatively charged oxygen atom; or    R′ is an oxygen atom and R″ represents either an oxygen atom or an oxygen atom carrying a protecting group; and            b) when x=0, R′ is an oxygen atom carrying a protecting group and R″ is either a hydrogen, halogen, or a di-substituted amine group.        
When x is equal to 1, R′ is an oxygen atom and R″ is an oxygen atom, the method is in this case the so-called phosphodiester method; when R″ is an oxygen atom carrying a protecting group, the method is in this case the so-called phosphotriester method.
When x is equal to 1, R′ is a hydrogen atom and R″ is a negatively charged oxygen atom, the method is known as the H-phosphonate method.
When x is equal to 0, R′ is an oxygen atom carrying a protecting group and R″ is a halogen, the method is known as the phosphite method, and when R″ is a leaving group of the disubstituted amine type, the method is known as the phosphoramidite method.
The conventional sequence used to prepare an oligonucleotide using reagents of the type of formula (I), basically follows four separate steps: (a) coupling a selected nucleoside which also has a protected hydroxy group, through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (e.g., the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (“deprotection”) from the now substrate-bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate-bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions, such as ammonium hydroxide, in a known manner.
The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides on a support by means of known chemistry. During array fabrication, different monomers can be deposited at different addresses on the substrate during any one iteration so that the different features of the completed array will have different desired polynucleotide sequences. The coupling can be performed by depositing drops of an activator and phosphoramidite at the specific desired feature locations for the array. One or more intermediate further steps can be required in each iteration, such as the conventional oxidation and washing steps.
The foregoing methods of preparing polynucleotides are well known and described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in “Synthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 5,153,319, 5,869,643, EP 0294196, and elsewhere. The phosphoramidite and phosphite triester approaches are most broadly used, but other approaches include the phosphodiester approach, the phosphotriester approach and the H-phosphonate approach. Such approaches are described in Beaucage et al., Tetrahedron (1992) 12:2223-2311. A more recent approach for synthesis of polynucleotides is described in U.S. Pat. No. 6,222,030 B1 to Dellinger et al., issued Apr. 24, 2001.
Although much research has been directed towards improving the feasibility and outcome of the various steps in the synthetic cycle, the basic strategy has not changed in the last 20 years. While the methodology would, therefore, appear to be rather well founded and time tested, new avenues of research in the blossoming fields of molecular biology and diagnostic medicine are continually pioneering novel applications of the technology. Along with these new applications has come an urgent demand for a synthetic method of much broader scope which will allow for the generation of high purity, long sequence DNA on unique and varied surfaces. Of equal gravity is the reality that the untold myriad of present and future applications of solid phase DNA synthesis will often be constrained by extreme physical parameters which are incongruent with the established (yet narrow) set of phosphoramidite compatible reagents and solvents. One such incompatibility is manifest as the unacceptable background fluorescence resulting from unwanted reagent interactions with the glass surfaces employed for many DNA microarrays. The source of the residual fluorescence is poorly understood but the ultimate result is loss of sensitivity in assays that rely on fluorescence reporting of DNA binding. The situation is utterly unacceptable in the field of diagnostic medicine where false negative or false positive results can wholly negate the benefit of the technology.
A second concern with the manufacture of DNA microarrays is that of the rapid, efficient, accurate, and reproducible synthesis of DNA features on derivatized glass substrates. The demands of large scale production of microarrays is forcing the industry to investigate new paradigms of allocating reagents on the substrates. One current method of delivering the reagents consists of jetting them out of inkjet print heads. While commercial grade inkjet heads have thus far sufficed for proof of concept pilot productions, growing requirements for speed and precision are now clearly demanding more efficient and reliable hardware. Unfortunately, the more attractive industrial inkjet heads, which would appear to offer all of the attributes wanting in their less robust counterparts, require highly viscous inks in order to operate.
While viscosity is not generally an insurmountable physical constraint, the problem becomes more apparent when one considers that there is a very limited set of solvents, which support phosphoramidite coupling to a satisfactory degree. Only highly polar, aprotic solvents support the reaction, but the list of candidates is most notably constrained by the requirement for >99% efficiency in each subsequent coupling step. Otherwise attractive solvents are of no use in an arena where the final oligomeric product must be devoid of any deletions, mutations, or other errors attributed to inefficient coupling. Most disconcerting is the fact that most solvents or solutes that can be expected to increase the viscosity of a useful solvent are either polymers (ethylene glycols) or polyols (i.e. glycerol) which either retard the reaction or act as substrates, which consume the phosphoramidite.
Therefore, there is a need in the art to address the aforementioned deficiencies and shortcomings.